Type 2 diabetes mellitus is a major cause of morbidity and mortality. In most instances, peripheral resistance to insulin's action in muscle and fat accounts for the elevated blood glucose seen in diabetics. The major goal of this research project is to identify and characterize the signaling pathways that are regulated by the insulin receptor. This will be accomplished initially by studying insulin regulation of proteins that contain src homology (SH) 2 and SH3 domains. The plan is to further characterize the pathways activated by phosphatidylinositol (PI) 3- kinase, Nck and Ras. P13-kinase and Ras are likely to play critical roles in coupling the insulin receptor to mitogenic signaling pathways and glucose transport. Nck is a promising candidate signaling molecule for involvement in insulin signaling, because recent evidence suggests that this molecule is recruited to the insulin-signaling pathway and therefore may perform a unique function in a central downstream pathway. Specific aims include: (l) To determine the mechanism of Ras activation by the insulin receptor. (IA) To determine the consequences of binding of the 6RB2-Sos complex to IRS-I or Shc in terms of activation of Ras by the insulin receptor, and to determine the mechanism whereby these interactions activate Ras. (1B) To determine how the insulin receptor activates Sos. (2) To determine the role of Nck in insulin signaling. Nck is thought to regulate signaling pathways by functioning as an adaptor molecule that couples IRS-I to a downstream signaling molecule bound to the SH3 domains of Nck. (2A) To determine the immediate downstream targets of Nck. We will identify the protein(s) that interact with Nck in vivo in insulin responsive tissues. (2B) Once downstream targets of Nck have been identified, their mode of regulation and their in vivo relevance to insulin signaling will be investigated. (2C) To directly test the role of Nck in mediating responses that are known to be regulated by insulin, for example mitogenesis and translocation of GLUT4 to the plasma membrane. If, as predicted, Nck is found to mediate some of these responses, the mechanism of this regulation will be determined. (2D) To determine the role of Nck in mediating the activation of the Ras-MAP kinase signaling pathway. (3) To clarify the role of PI3-kinase in insulin signaling. Genetic evidence in lower organisms and experimental studies using PI3-kinase inhibitors in mammalian cells have suggested that PI3 kinase may be important in linking the insulin receptor to activation of p7056K and to glucose uptake and GLUT4 mobilization to the plasma membrane. Using more definitive experimental approaches we will set out (3A) To determine whether P13-kinase mediates (a) glucose uptake and (b) GLUT4 translocation to the PM in two insulin responsive tissues (fat and muscle). (3B) To study the mechanism whereby PI3-kinase mediates its effect in insulin-stimulated cells. (3C) To determine whether PI3-kinase couples the insulin receptor to activation of 70s6k.